Characterizing Large-Scale Receptor Clustering on the Single Cell Level: A Comparative Plasmon Coupling and...

Area of Science:

• Biophysics
• Cell Biology
• Nanotechnology

Background:

• Spatial clustering of cell membrane receptors, like epidermal growth factor receptor (EGFR), is crucial for signal initiation.
• Receptor distribution heterogeneity on the cell surface holds potential as a diagnostic biomarker.
• High-throughput, scalable assays are needed to map receptor clustering for diagnostic applications.

Purpose of the Study:

• To develop and validate a high-throughput imaging method for mapping large-scale EGFR clustering.
• To compare the efficacy of hyperspectral plasmon coupling microscopy with gold nanoparticle labels against direct stochastic optical reconstruction microscopy (dSTORM).
• To investigate the relationship between EGFR cluster size, activation status, and underlying cellular structures.

Main Methods:

• Utilized gold nanoparticles (NPs) as bright labels for EGFR in hyperspectral plasmon coupling microscopy.
• Employed direct stochastic optical reconstruction microscopy (dSTORM) for high-resolution EGFR cluster analysis.
• Analyzed changes in NP interparticle separation and spectral shifts to infer clustering dynamics.

Main Results:

• dSTORM revealed average EGFR cluster sizes of 172 ± 99 nm (MDA-MB-468) and 150 ± 90 nm (HeLa), decreasing upon activation.
• Hyperspectral imaging showed that EGFR cluster size differences correlate with NP interparticle separations and spectral shifts.
• Hyperspectral plasmon coupling microscopy identified similar EGFR clustering trends as dSTORM, but significantly faster.

Conclusions:

• Hyperspectral plasmon coupling microscopy with NP labels offers a rapid, high-throughput alternative for mapping EGFR clustering.
• EGFR cluster sizes are influenced by activation status and the cortical actin network.
• This technique has potential for diagnostic biomarker development by analyzing spatial receptor heterogeneity.